Field of the Invention
The disclosure relates to the field of molecular biology, and in particular to a cleaved Deoxyribonucleic acid (DNA) detection method, a DNA fragment detection kit and the use thereof.
Background
Scientific researchers need to extract DNA from different tissue samples in the molecular biology research. Wherein, the body fluid contains the fragmented DNAs, and the detection of the fragmented DNA is an important step for the molecular biology research. For example, the plasma DNA, also called as the circulating DNA, is the extracellular DNA in the blood, of which the length is tens to hundreds of nucleotides (the main peak is approximately 167 bp); the plasma DNA can exist in the form of DNA-protein complex, and also can be free DNA fragments. The plasma DNA is derived from the DNA release of a small amount of aging dead cells. The generation and removal of the plasma DNA are in a dynamic balance status, and are maintained in a relatively constant low level; 1 mL of the plasma approximately includes the amount of 2000 genomic DNA, namely, the content is extremely low, thus the gene information cannot be detected by adopting the Polymerase Chain Reaction (PCR) in the prior art.
In addition, the source of fresh tissue samples is limited, the Formalin Fixed Paraffin Embedded (FFPE) method solves the problem that the fresh tissues are hard to be stored for a long time, but during the preparation and storage process of the FFPE tissue samples, the tissue is fixed by formalin and embedded by paraffin which is easy to cause cross-linking and fragmentation of the DNA, which is 100 bp-3000 bp fragments, the scientific researchers are hard to obtain enough DNA samples with high quality for high-sensitive detection. At present, a large amount of samples in the world are processed by the FFPE method, and the tissue samples have become one of the most normal biological resources for the scientific research.
The traditional method of detecting whether the DNA fragment has base mutation or other mutations mainly implements detection by implementing PCR amplification for the specific test region. The traditional PCR technology usually adopts a pair of primers which cross two sides of the target region, takes the DNA as a template, which is a method for in vitro enzymatic synthesis of specific DNA fragments. high temperature denaturation, low temperature annealing (renaturation), optimum temperature extension and the like form a period, which circularly operate, so as to make the target DNA be rapidly amplified; the technology has characteristic of high specificity, high sensitivity, simple operations, time-saving property and the like, and is widely applied in fundamental research of gene isolation, cloning and nucleotide sequence analysis, and the diagnosis of diseases. But the PCR technology requires that the DNA template between a pair of primers has no fragmentation point, which provides a great challenge for the seriously-fragmented DNA templates. As the traditional PCR amplification requires that the amplified region is kept complete, the traditional method has low detection sensitivity for the DNA fragment, which limits the application thereof in detecting the DNA fragment samples.